This is an R21 exploratory/developmental proposal. The goal is to investigate the feasibility of employing novel light sensitive amphetamine-like molecules as photoaffinity labels to identify and characterize amphetamine-related binding sites. This goal will be achieved through chemical synthesis and biochemical analysis. The emphasis will be to synthesize compounds known as aminoalkylphenones, The new compounds will be evaluated for their functional similarities to amphetamine in affecting monoamine neurotransmitter uptake and release and will be used to identify amphetamine-related binding sites using photoaffinity labeling techniques and MALDI-TOF analysis. The information generated will provide insight into the structural requirements for binding and efficacy of amphetamine-like molecules at the monoamine uptake transporters and may identify other amphetamine binding proteins that would not be discovered using other approaches. Ultimately, this research will aid in the rational design of therapeutic agents with greater specificity for the treatment of drug abuse, depression, and other psychiatric diseases associated with perturbed monoamine homeostasis and may also identify new biological targets for pharmacological intervention. The specific aims are: 1) To design, synthesize, and physically characterize novel amphetamine-related drugs belonging to a chemical class known as aminoalkylphenones. Compounds with various substituents on the phenyl ring and on the alkylamine side-chain will be synthesized for pharmacological evaluation. When chiral molecules are possible, stereoisomers will either be individually synthesized using enantioselective syntheses or racemates will be resolved using fractional crystallization or chiral chromatography. Radiolabeled variants will be synthesized as potential photoaffinity labels or radiotracers. All new compounds will be characterized by physical and chemical methods. 2) To generate pharmacological profiles of the newly synthesized compounds in vitro. Because inhibition of monoamine uptake and stimulation of monoamine release are major mechanisms of action for amphetamine-like drugs, the new compounds will be tested for their abilities to inhibit neurotransmitter uptake into cells stably expressing the cloned human monoamine transporters and they will be evaluated for their abilities to evoke the release of stored monoamine neurotransmitters under superfusion conditions. 3) To identify and biochemically characterize binding sites for new amphetamine-related photoaffinity labels. The photoaffinity labels synthesized under Specific Aim 1 will be used to identify binding domains and contact residues for amphetamine-like molecules within monoamine transporters or other protein binding sites. After photoaffinity labeling of cultured cells or native tissues, 2-D PAGE, MALDI-TOF analysis, limited proteolysis, N-terminal sequencing, and radiosequencing of the purified, protectably labeled proteins will identify domains and individual amino acids that form the drug acceptor sites.